Dietary supplementation of a Lactobacillus postbiotic modulates acute stress responses and impacts temporal variation in the gut microbiome of dogs

Technical Summary: Dietary Supplementation of a Lactobacillus Postbiotic Modulates Acute Stress Responses and Impacts Temporal Variation in the Gut Microbiome of Dogs

Problem Statement
Stress and anxiety, particularly noise phobias, are prevalent issues in companion dogs, often leading to welfare concerns and owner distress. While pharmaceutical interventions exist, they carry limitations regarding side effects and owner preference for natural solutions. Emerging evidence suggests the gut-brain axis plays a critical role in modulating stress responses, yet clinical data on postbiotics (inanimate microorganisms or fragments conferring health benefits) for stress management in dogs remains scarce. Previous studies in mice indicated that a specific Lactobacillus postbiotic (LBHF) could reduce stress markers and alter the microbiome, but these findings required validation in a canine model.

Methodology
The study employed a randomized, placebo-controlled, investigator-blinded parallel-group intervention trial involving 24 healthy Beagle dogs selected for pronounced cortisol reactivity to stress.

• Intervention: Dogs received either a Lactobacillus postbiotic (LBHF; 300 mg/day) or a placebo (microcrystalline cellulose) for 28 days, administered via treats alongside a standard kibble diet.
• Stress Challenges: Two distinct stress models were utilized at baseline (days -5 and -4) and post-treatment (days 29 and 30):
  1. Thunderstorm Simulation: A 9-minute session in an open-field room with a 3-minute digital thunderstorm playback (82–94 dB).
  2. Car Ride: A 10-minute transport stress test in a secured crate within a vehicle.
• Endpoints:
  • Primary: Plasma cortisol levels (pre- and post-challenge).
  • Secondary: Heart rate (HR) reactivity, behavioral stress scores, fecal Bristol Stool Scale (BSS) scores, and temporal stability of the gut microbiome.
  • Omics: Shotgun metagenomics (fecal) and semi-targeted metabolomics (plasma and fecal) were performed to assess community composition and metabolic shifts.
• Dietary Challenge: Following the final stress test, all dogs were switched to a canned food diet for one week to induce microbiome shifts and test resilience.
• Statistical Analysis: Generalized additive models (GAMs) and linear mixed models were used to assess treatment effects on physiological and behavioral endpoints. Multivariate analyses (PERMANOVA, Principal Response Curves, Tensor Factorization, MEFISTO) were applied to microbiome and metabolome data to evaluate temporal stability and covariation.

Key Results

• Physiological Stress Response:
  • Heart Rate: LBHF supplementation significantly reduced HR reactivity during the thunderstorm challenge compared to placebo, with an average reduction of ~20.6 beats per minute (P=0.021). No significant difference was observed in HR reactivity during the car ride challenge.
  • Cortisol: While LBHF tended to decrease cortisol responses during the thunderstorm challenge, the reduction was not statistically significant (P=0.18). No differences were found in the car ride test.
  • Behavior: No significant between-group differences were observed in behavioral stress scores for either challenge. However, a positive association was confirmed between ranked behavioral stress scores and HR, validating HR as a physiological marker of acute stress.
• Gut Health and Transit:
  • LBHF treatment led to significantly lower fecal Bristol Stool Scale (BSS) scores (indicating firmer stools) during the diet challenge phase (P=0.03), suggesting improved transit time and stability in response to dietary changes.
• Microbiome Dynamics:
  • LBHF treatment significantly impacted the temporal variation of the gut microbiome. Treated dogs exhibited reduced intragroup variation (beta dispersion) and greater temporal stability in microbial community composition compared to the placebo group, particularly during the dietary intervention.
  • While overall alpha diversity was not significantly different between groups, specific taxonomic shifts were observed. The LBHF group showed a decrease in several Bifidobacterium species and Fusobacterium, alongside an increase in taxa such as Sutterella, Anaerobiospirillum, and a novel CALVGN01 species.
• Metabolomics:
  • Plasma: LBHF treatment was associated with an overall increase in circulating tryptophan and other amino acids. Stress challenges in the post-treatment phase altered the metabolism of tryptophan, shifting it toward the serotonin pathway, though LBHF did not significantly alter the specific ratios of serotonin to tryptophan or kynurenine.
  • Fecal: Multivariate analysis identified a specific factor (C17) related to LBHF treatment, characterized by changes in triglyceride concentrations.
  • Integration: Multi-omic integration revealed that specific latent factors derived from fecal microbiome and metabolome data (specifically Factors 4, 8, and 9) were predictive of the reduction in HR reactivity, suggesting a mechanistic link between microbial/metabolic shifts and stress attenuation.

Significance and Claims
The paper concludes that dietary supplementation with the Lactobacillus postbiotic (LBHF) can attenuate physiological stress responses in dogs, specifically by reducing heart rate reactivity during acute, noise-induced stress. The study supports the hypothesis that postbiotics act via the gut-brain axis, evidenced by:

1. Stress Modulation: A cortisol-independent calming effect observed through reduced heart rate reactivity.
2. Microbiome Stabilization: The ability of LBHF to enhance the temporal stability of the gut microbiome and reduce community variation, which may contribute to resilience against stress and dietary perturbations.
3. Metabolic Impact: Modulation of circulating amino acids (specifically tryptophan) and fecal metabolites that correlate with stress responses.

The authors posit that these findings support the use of postbiotics as a natural, non-pharmaceutical means of managing stress and anxiety in dogs, offering a stable alternative to probiotics with demonstrated benefits for gut transit time and microbial community stability. The study acknowledges that while the anxiolytic effect was clear in the thunderstorm model, it was not replicated in the car ride model, potentially due to differences in stress acuity or anticipatory effects.